High speed flow cytometer droplet formation system and method

ABSTRACT

A droplet forming flow cytometer system allows high speed processing without the need for high oscillator drive powers through the inclusion of an oscillator or piezoelectric crystal such as within the nozzle volume or otherwise unidirectionally coupled to the sheath fluid. The nozzle container continuously converges so as to amplify unidirectional oscillations which are transmitted as pressure waves through the nozzle volume to the nozzle exit so as to form droplets from the fluid jet. The oscillator is directionally isolated so as to avoid moving the entire nozzle container so as to create only pressure waves within the sheath fluid. A variation in substance concentration is achieved through a movable substance introduction port which is positioned within a convergence zone to vary the relative concentration of substance to sheath fluid while still maintaining optimal laminar flow conditions. This variation may be automatically controlled through a sensor and controller configuration. A replaceable tip design is also provided whereby the ceramic nozzle tip is positioned within an edge insert in the nozzle body so as to smoothly transition from nozzle body to nozzle tip. The nozzle tip is sealed against its outer surface to the nozzle body so it may be removable for cleaning or replacement.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of a contractawarded by the Department of Energy.

This application is a continuation of application Ser. No. 08/323,270,filed Oct. 14, 1994, now abandoned.

BACKGROUND OF THE INVENTION

Generally this invention relates to droplet flow cytometers such as areused for the analysis and sorting of substances contained withinseparate droplets. Specifically, the invention relates to aspects ofsuch systems which act to form regular droplets after exit from a nozzleorifice.

Droplet flow cytometers have been in clinical and research use for manyyears. Basically, the systems act to position small amounts of asubstance within individual droplets of a sheath fluid. These dropletscan be made uniform by utilizing an oscillator which emits a predominantfrequency. These oscillations are usually applied to the nozzlecontainer. Since droplet flow cytometry is heavily utilized in bothresearch and clinical environments, such systems have been the subjectof much refinement. One of the facets of these systems which has beenparticularly challenging, however, is the aspect of controlling the dropformation. As to this aspect it has not only been difficult topractically achieve processing rates of much more than 40 kilohertz, ithas also been difficult to deal with the incidents of using relativelyhigh power to drive the oscillators involved.

It should be noted that each of the challenges faced in the field ofdroplet formation for flow cytometers is largely unique to that field.Even seemingly similar fields such as those involving channel-type flowcytometers are not very analogous as they do not face such problems.Their operation as continuous flow devices rather than droplet formationdevices makes much of the understandings available in that fieldinapplicable to the challenges and problems faced in flow cytometrydroplet formation systems.

To some degree the challenges for droplet formation may be the result ofthe fact that although drop formation has been modeled with significanttheoretical detail, in practice it still remains a somewhat empiricalsubject. While on one level exhaustive mathematical predictions arepossible, in practice these predictions can be greatly tempered--and areoften revised--by the fact that materials limitations, inherentsubstance variations, and the like contribute heavily to the end result.A number of "advances" in this field have even proved to be eitherunnecessary or unworkable in practice.

The level of oscillation energy required in order to achieve uniformdroplet formation has, prior to the present invention, been very subjectto empirical constraints. This power (often expressed as a voltageamplitude applied to a piezoelectric crystal oscillator) has previouslybeen in the ten volt range. Unfortunately, this relatively high voltagenot only results in a need for more robust circuitry, but it also hasthe undesirable practical consequence of resulting in undesirableelectromagnetic emissions. These emissions can impact the sensitivity ofthe flow cytometer or other nearby equipment. Further, as the desire forhigher processing frequencies is pursued, this problem is compounded.Although these problems have been know for years, prior to the presentinvention it has apparently been an accepted attitude that in order toachieve higher frequencies, still higher oscillation energies are aphysical requirement. This invention proves this expectation to beuntrue. An example of the extremes to which this rational had beenapplied is shown in U.S. Pat. No. 4,361,400 to Gray where dropletformation frequencies in the range of 300 to 800 kilohertz had beenachieved. This design had required an oscillator powered byapproximately 80 volts. The apparent physical requirement of higherpowers in order to achieve higher droplet frequencies may have been onereason that most practical droplet flow cytometers operated only in therange of 10 to 50 kHz. The present invention shows that such arelationship is not a physical requirement and, in fact, shows thatdroplet formation speeds in the 100-200 kHz range are actually possiblewith only millivolts of power applied to an oscillator.

Yet another problem practically encountered in this field was thechallenge of resonances existing within the nozzle assembly. Again, thisappears to have simply been accepted as a necessary incident of workablesystems and may have resulted in an attitude among those having ordinaryskill in the art that it was not practical to vary frequency withoutunacceptable changes in the performance of the entire system. There alsoseems to have been some confusion as to the appropriate way to apply thedroplet forming oscillations. U.S. Pat. No. 4,302,166 shows that theoscillations are applied to the nozzle container perpendicular to thefluid flow, whereas, U.S. Pat. No. 4,361,400 suggests applying theoscillations to the nozzle container parallel to the lines of flow. Infact, the present invention discloses that each of these systems aresuboptimal in that they may even act to generate the resonances andvariations in frequency response of the nozzle system.

An even more paradoxical situation exists with respect to the problem ofmaintaining laminar flow within the nozzle system of a droplet flowcytometer. Although those having ordinary skill in this field have knownfor years that maintaining laminar flow was desirable, until the presentinvention, practical systems utilizing replacement tips have not beenoptimally designed so as to achieve the goal of truly laminar flow. Forinstance, U.S. Pat. No. 4,361,400 as well as the 1992 publication bySpringer Laboratory entitled "Flow Cytometry And Cell Sorting", eachshow replaceable nozzle tip designs in which laminar flow is disruptedat the junction between the nozzle body and the nozzle tip. Again, suchdesigns seem to present almost a paradox in that they obviously are notoptimum from perspective of a goal which has long been known as thosehaving ordinary skill in the art. The present invention not onlyrecognizes this goal but also demonstrates that a solution has beenreadily available.

Yet another problem encountered in this field is the need to varyparameters to optimize actual conditions encountered in processing.Again theory and practice did not mix well. While systems were usuallydesigned for optimum conditions, in actual usage such conditions rarelyexisted. Thus, as U.S. Pat. No. 4,070,617 recognized, designs whichallow variation of the substance output velocity within the sheath fluidwere desirable. Although such systems permitted some variation, it wasrecognized that such variations necessarily made conditions within theflow cytometer suboptimal for the simple reason that there is a verydefinite physical relationship between the sheath substance and dropparameters which must be maintained. Since these parameters are wellknown to those having ordinary skill in the art (as also indicated inU.S. Pat. No. 4,302,166), the variations required in practice appear tohave been accepted as a necessary evil. To some extent, the resultingreduced resolution appears to have been accepted without question.Again, the present invention realizes that approaches which movedconditions away from optimal were not a necessary incident of adaptingto conditions practically encountered; it shows that solutions whichallow for variation and yet maintain optimal flow conditions arepossible.

As explained, most of the foregoing problems had long been recognized bythose having ordinary skill in the art. Solutions, however, had eitherbeen perceived as unlikely or not been recognized even though theimplementing elements had long been available. This may also have beendue to the fact that those having ordinary skill in the art may not havefully appreciated the nature of the problem or may have been due to anactual misunderstanding of the physical mechanisms involved. Theseappear to have included the misunderstanding that actually moving thenozzle was the proper way to induce the droplet forming oscillations andthe simple failure to realize that it was possible to coordinate thedesire for replaceable nozzle tips with the desire for laminar flowwithin the flow cytometer nozzle assembly. Similarly, those skilled inthe art had long attempted to achieve higher frequency systems whichwere practically implementable and had attempted to achieve variationswhich would to the largest extent possible maintain optimal conditions.Their attempts often led them away from the technical directions takenby the present invention and may even have resulted in the achievementsof the present invention being considered an unexpected result of theapproach taken.

SUMMARY OF THE INVENTION

The present invention involves a number of improvements which areapplicable to a flow cytometer droplet system. These improvements eachoffer independent advantages and may be combined synergistically toproduce a great increase in the performance of droplet flow cytometers.The preferred embodiment involves a piezoelectric oscillator containedwithin the sheath fluid above a continuously converging nozzlecontainer. This nozzle container acts to amplify the oscillations whichare directly and directionally coupled to the sheath fluid. Further, thelocation of the substance introduction tube may be adjusted within aconvergence zone so as to vary the rate at which the substance isintroduced relative to the rate at which the sheath fluid is introducedto maintain optimal conditions. In addition, a replaceable nozzle tip isfit within an edge insert and sealed on its outer surface so as tomaintain laminar flow and enhance the amplification of the oscillationthroughout the converging nozzle body. As a result of the combination ofthese various features, the present invention not only achievespractical processing at frequencies of many multiples of typical priorart devices, but it also achieves these processing rates at oscillationpowers which are several orders of magnitude less than those typicallyutilized.

Accordingly, one of the objects of the invention is to provide for a lowpower system which allows high processing rates. In keeping with thisobject, one goal is to achieve direct coupling of the oscillations tothe sheath fluid and thus minimize any losses associated with typicalmaterial interfaces. In keeping with this object, another goal is toprovide for a system which actually amplifies the oscillations so as toproduce acceptable fluid variations at the nozzle tip.

Yet another object of the invention is to minimize the impacts ofresonance frequencies within the nozzle system. In keeping with thisobject, a goal is to directionally couple the oscillations to the sheathfluid. It is also a goal to isolate the oscillations from imparting uponthe sheath fluid in more than the desired direction.

A further goal of the invention is to provide for a system which allowsfor the maintenance of laminar flow within the entire nozzle assemblywhile allowing for both replaceable nozzle tips and for internalvariations. The present invention achieves the first object by providinga design which avoids the unnecessary impacts of a seal on the flowcondition within the nozzle container. The second object is achieved byproviding a system which varies the location at which a substance isintroduced while still maintaining optimal, laminar conditions.

Still another object of the invention is to provide for a practicallyimplementable system. In keeping with this object, one goal is toprovide a system which can be easily cleaned and for which componentscan be easily replaced. A goal is also providing a design which can berelatively easily and inexpensively manufactured.

Naturally, further objects of the invention are disclosed throughoutother areas of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross sectional view of an embodiment of theinvention showing the various features combined.

FIG. 2 is a plot of the droplet onset energy of prior art designscompared to that of the present invention.

FIG. 3 is a schematic cross sectional view of an alternative designshowing the automatic substance adjustment feature and a directionallycoupled, external oscillator.

FIG. 4 is a cross sectional view of a replaceable tip design accordingto one embodiment of the invention.

FIG. 5 is a cross sectional view of a prior art replaceable nozzle tipdesign.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As mentioned, the present invention involves an improved flow cytometerdroplet nozzle system which incorporates a variety of features. As shownin FIG. 1, the flow cytometer system (1) involves nozzle container (2)which establishes nozzle volume (3). Nozzle volume (3) is supplied aliquid by sheath fluid port (4) which acts to introduce a sheath fluidfrom some sheath reservoir (5). During operation, the sheath fluid flowsthrough nozzle container (2) and out nozzle exit (6) into free fall area(7).

Since the sheath fluid is typically an unreactive substance such as asaline fluid and is an analytically transparent, it has introducedwithin it some desirable substance such as cells or parts of cells orother items. This substance is maintained in substance reservoir (8) andis introduced to nozzle volume (3) through substance introduction port(9). Through hydrodynamic focusing, the substance flows and is separatedinto single cell units within the sheath fluid and exits at nozzle exit(6).

In order to form regular droplets, the preferred embodiment utilizes apiezoelectric crystal (10) to cause oscillations (11) within the sheathfluid. These oscillations are transmitted as pressure variations throughto nozzle exit (6) and act to allow jet (12) to form regular droplets(13) through the action of surface tension. These processes are wellunderstood and are further explained in a number of references includingthe 1992 reference entitled "Flow Cytometry and Cell Sorting" by A.Radbruch (©Springer-Verlag Berlin Heidelberg) and the 1985 referenceentitled "Flow Cytometry: Instrumentation and Data Analysis" edited byMarvin A. Van Dilla, et al. (®Academic Press Inc. (London) Ltd.) each ofwhich are incorporated by reference.

As shown in FIG. 1, one of the features of the preferred embodiment isthe location of piezoelectric crystal (10) within nozzle volume 3. Bythis feature the oscillator acts to initiate oscillations (11) withinthe nozzle volume. The oscillator thus may be directly coupled to thesheath fluid. These oscillations are transmitted through the sheathfluid as it flows out nozzle exit (6) and forms droplets (13) belownozzle (6) in freefall area (7). Naturally, although shown to bedirectly below it is possible that the nozzle assembly could be orientedon its side or in some other relationships and so droplets (13) mightform at some other location and yet still be characterized as "below"nozzle tip (6) since they will form in the direction that jet (12) isemitted from nozzle exit (6).

As is well understood, by allowing sheath fluid and the substance toexit from nozzle container (2), cells or cell fragments may be isolatedin singular fashion within separate droplets (13) for analysis by sensor(14) which feeds its information to analysis equipment (15). Analysisequipment (15) may provide the necessary data or may act to furtherprocess droplets (13) through some equipment such as an electrode innozzle volume (3) in combination with sorting electrostatic fieldequipment (16) as is well known in the art. When electrostaticpotentials are applied, they may be applied differentially to eachdroplet based upon the delay in droplet formation. This analysisequipment (15) may also include a separate laser which inducesfluorescence and the like in specific cells to allow further sensing andfacilitate conducting analysis as well.

As may be easily understood from FIG. 1, this type of flow cytometer, adroplet flow cytometer, operates quite differently from a channelforming flow cytometer. In channel-type flow cytometers, oscillators andthe theories involved are not relevant as no freefall or dropletformation is required. Further, while the nozzle exit orifice isapproximately 50 to 150 microns in diameter in droplet forming flowcytometers, in channel-type flow cytometers, the orifice can be muchlarger--on the order of 1000 microns. This causes extremely differentconditions and has resulted in the two fields being treated somewhatdifferently by those involved.

Another feature of the invention is how the oscillator couples toactually cause the formation of droplets (13). As shown in FIG. 1, theoscillator is in this embodiment piezoelectric crystal (10). While,naturally, a variety of different devices could be used in order toachieve oscillation (11), by using piezoelectric crystal (10) a host ofdifferent frequencies and powers are possible. It should be understood,however, that while the use of some piezoelectric crystal is usually thepreferred technique, the invention should not be considered as limitedto that type of oscillator as its teachings can be broadly applied.

As shown in FIG. 1, piezoelectric crystal (10) is configured as aring-shaped crystal which occupies most of the top end of nozzlecontainer (2). This ring may be mounted directly to nozzle container (2)in a manner so as to be situated not wholly outside of the nozzle volume(3). It need not vibrate the nozzle container and, indeed is designed toavoid it. Its oscillations (11) may also be made to occur generally in adirection parallel to the central axis of nozzle container (2) as shown.Further, these oscillations (11) are essentially coupled to the sheathfluid, not to the nozzle container. Thus, rather than taking thedirections suggested by some of the prior art involving moving theactual nozzle container, the present invention acts unidirectionallyupon the sheath fluid to cause pressure variations within the sheathfluid. These pressure variations move down nozzle volume (3) and mayactually be amplified by the shape of nozzle container (2) so as tocause surface tensions variations in jet (12) as it emerges from nozzleexit (6). These variations act to pinch off jet (12) and thus formdroplets (13). Since the sheath fluid is not substantially compressible,these pressure variations may pass relatively unattenuated and in factmay be amplified through nozzle volume (3) to achieve the desireddroplet formation effect. While others may have considered the desire tocoupling directly to the sheath fluid, they failed to recognize ways todo this and did not recognize that they could have positioned theoscillator within the sheath fluid for most efficient coupling.

Both the direct coupling of oscillations (11) to the sheath fluid andthe directional nature of the oscillations (11) contribute to theinvention's ability to achieve droplet formation at power levels whichare several orders of magnitude less than those of the prior art. As maybe understood from FIG. 1, piezoelectric crystal (10) may directlytransfer the vast majority of its energy to the sheath fluid. To furtherenhance the transfer of the majority of the energy into the sheath fluid(rather than the nozzle as often suggested by the prior art), theinvention may also incorporate the designing of massive nozzle containerelements so as to minimize the transfer of energy through theseelements. As may be easily understood, by positioning the oscillatorwithin the sheath fluid, frequency dependencies and resonances which arecaused by the vibration of the entire nozzle container can be greatlyreduced. Thus, contrary to the teachings of the prior art whichsuggested vibrating the entire nozzle container, the present inventioncan specifically avoid such vibrations. This acts to avoid resonancefrequencies as might occur through vibrations perpendicular to the linesof flow which may be inevitable whenever the entire nozzle assembly isvibrated. Contrary to those teachings which have suggested mounting theentire nozzle assembly on a flexible membrane so as to allow the entirenozzle assembly to move, the present invention relies not on movement ofnozzle container (2) but rather on pressure waves within the sheathfluid in nozzle volume (3). This aspect greatly reduces the amount ofpower necessary to cause droplet formation and greatly reduces theappearance of resonance frequencies which occur as a result of theentire vibration of nozzle container (2) among other aspects.

Referring to FIG. 2, the dramatic impact of these reductions can beunderstood. FIG. 2 shows a conceptual plot of the rough energy ofdroplet formation onset versus frequency anticipated for the presentinvention. As shown in FIG. 2, the energy (expressed in terms of voltsapplied to a given piezoelectric crystal) is reduced by orders ofmagnitude. This reduction has been demonstrated for a number offrequencies. As shown in FIG. 2, the prior art which typically operatedin the 10 volt range now only requires ten millivolts or so.

In addition, as shown in FIG. 2, it can be seen that the prior art wasalso subject to a great number of resonance frequency variations (shownby the peaks and valleys in the plot of the prior art). These peaks andvalleys were to a large extent caused not only by the amount of powerrequired but also by designs which were based upon movement of theentire nozzle assembly rather than merely pressure waves within thenozzle assembly. In sharp contrast to the prior art characteristicconceptually shown in FIG. 2, the present invention not only achievesdroplet formation with dramatically lower voltages but it also achievesthese levels over a relatively large frequency range with very smallresonance variations compared to those of the prior art. These relativeplots are believed to represent significant differences in resultbetween prior art designs and those of the present invention. Whilenaturally variations will occur due to the particular nozzle designsultimately chosen, it is believed that through the teachings of thepresent invention these dramatic variations should be practicallyachievable in many cases.

Referring again to FIG. 1, it can be seen that besides merelypositioning the oscillator within nozzle volume (3), the embodiment alsois designed to minimize the number of material interfaces through whichthe oscillations must pass before being imparted upon the sheath fluid.While, naturally, it would be possible to position piezoelectric crystal(10) directly exposed to the sheath fluid, for contamination and otherreasons, the preferred embodiment allows for the inclusion of protectivecoating (17) over piezoelectric crystal (10). This protective coating(17) may actually be some type of epoxy or other coating which has notendency to interfere either with the sheath material or theoscillations (11) of piezoelectric crystal (10). Again, contrary to theteachings of the prior art which involve numerous material interfacesbetween the oscillator and the sheath fluid, the present inventionminimizes the number of material interfaces through which oscillations(11) must pass. Since any change in material can cause reflection andenergy losses, the preferred design allows for only one interfacematerial such as protective coating (17). Thus, only one interfacematerial exists between oscillator surface (18) and the sheath fluid. Bypositioning piezoelectric crystal (10) within nozzle volume (3) not onlycan the interface material be limited to the simple epoxy coatingmentioned, but also, the oscillator surface (18) can be positioned so asto face directly to the sheath fluid.

As mentioned, another aspect which helps the invention achieve itsextraordinary reduction in oscillation drive power is the fact that theoscillator is directionally coupled to the sheath fluid. In order toavoid resonances and energy transmissions in other than the desireddirection, the present invention recognizes that unidirectional couplingis desirable. In order to achieve this, as shown in FIG. 1 theembodiment provides for positioning piezoelectric crystal (10) so thatit is detached from the sides of nozzle container (2). Since allpiezoelectric crystals act in a manner so as to conserve volume duringoscillations, this avoids coupling the inherent perpendicularoscillations to nozzle container (2). Again, through this recognition,the invention can achieve a uniform pressure wave within the sheathfluid. Since oscillator surface (18) is oriented perpendicular to theprimary flow direction, the oscillations (11) are coupled substantiallyonly as a flow direction deemed to be primary, whether the average flowdirection, a specific location's flow direction, or even the directionat the nozzle exit (6). This allows for the oscillations to beunidirectionally applied to the sheath fluid and also aids in thereduction of resonance frequencies. As shown in FIGS. 1 and 3, thisunidirectional coupling can be achieved through the inclusion of adirectional isolator (19). As shown in FIG. 3, directional isolator (19)may be a separate element such as a rubber or other material which doesnot transmit frequencies of the predominant oscillation frequency. Asshown in FIG. 1, the directional isolator (19) may actually be spacer(20). Spacer (20) may be a separate element or, as shown in FIG. 1, maybe an integral portion of the top or cap of nozzle container (2) so asto simply act to space oscillator side (21) away from nozzle container(2). The unidirectional coupling of oscillations (11) to the sheathfluid may be enhanced by making oscillator surface (13) planar as shownin FIG. 1 and by making it cover most of the top surface area. Theoscillator is thus established substantially throughout a perpendicularcross sectional area (perpendicular to the primary flow direction) andwill cause oscillations throughout it. Thus the ring shaped crystaldesign coordinates the desire to maximize the surface area of oscillatorsurface (18) with the unidirectional desire by making it match thetypically circular cross section of nozzle container (2). Naturallyother shapes can also be used. Further, the coupling, shown in FIG. 1and in FIG. 3 as the portion of the top section of the nozzle container(2) may also be planar and may also be coupled along only one plane.These each contribute to making the main oscillation area cause only onedirection of oscillation as can be easily understood.

To further enhance the reduction in oscillation power achievable throughthe present invention, nozzle container 2 is also designed as acontinuously converging nozzle container. This acts to not only maintainlaminar flow throughout nozzle volume (3), but also to effectivelyamplify oscillations (11) as they travel in pressure waves through thesheath fluids from piezoelectric crystal (10) to nozzle exit (6). As maybe understood from FIG. 1, by continuously converging it is not meantthat nozzle volume (3) must constantly or uniformly converge throughoutits length, rather, it need only converge at all locations. Thus, nozzlevolume (3) has a largest cross-sectional area located at or near its topand has continuously diminishing cross-sectional areas along its lengththrough to nozzle exit (6).

Having a continuously converging nozzle container also helps inmaintaining laminar flow up to nozzle exit (6). In this regard nozzleexit (6) should be understood to exist not only at the actual endlocation of the orifice but more accurately at the point at which thereis a significant increase in the pressure gradient so as to make changesin the angle of convergence less important. Unlike the teachings of theprior art which frequently involve straight cylindrical sections withinnozzle container (2), this aspect of the invention specifically avoidssuch possibilities. This is somewhat surprising and may be treated withskepticism by those of ordinary skill in the art because traditionaltheories provide that once laminar flow is established such flow shouldcontinue in most applications when the nozzle container does not expandsharply. In contrast, this aspect of the invention suggests otherwise.While these traditional laminar flow theories may be appropriate in someinstances, the continuous convergence of the sheath fluid appearsdesirable in most droplet flow cytometers. To some extent this may bedue to the fact that the required acceleration of the sheath fluid andpressure, and the resulting increase in the friction of the sheath fluidagainst nozzle container (2), each make a constant convergence desirableto avoid nonlaminar flow results. Basically it has been empiricallyfound that through a continuously converging nozzle container optimalconditions for maintaining laminar flow can be created.

In addition to the aspect of maintaining laminar flow, the continuouslyconverging nozzle container can provide amplification of theoscillations (11). Similar to horn and other designs, the continuousconvergence combines with the principals of conservation of energy sothat the amplitude of the oscillations actually increases as it passesfrom piezoelectric crystal (10) to nozzle exit (6). This amplificationmay be maximized not only by positioning the oscillator at or near thelargest cross-sectional area but also by making oscillator surface (18)to have an area substantially as large as the largest cross-sectionalarea. In this regard by "substantially" it is meant that the oscillatorshould be as large as practically possible after consideration of thetypical desire to introduce substance through the center axis of nozzlevolume (3) as well as this invention's unique desire to maintainoscillator side (21) spaced apart from nozzle container (2). Theamplification may also be enhanced by providing for continuousconvergence from sheath fluid port (4) through to nozzle exit (6). Asmentioned earlier, each of the foregoing aspects also contribute to thepresent invention's extraordinary reduction in input power requirements.

To create oscillations (11), piezoelectric crystal (10) is poweredthrough an alternating voltage source (22) as those skilled in the artcan easily understand. Through the teachings of the present invention,alternating voltage source (22) may be configured to stimulate theoscillator with the voltage amplitude of less than 100 millivolts andthus represents orders of magnitude of reduction in the typical voltageapplied to piezoelectric crystals in such systems. This voltage may begreater than 10 millivolts or so as that was a representative level atwhich droplet formation seems to occur. It should be understood,however, that this limitation should not be taken as a lower limit sincethe teachings of this invention may become refined and alternativedesigns may be developed which result in further reduction in power.

Yet another independent feature of the present invention is its designto allow the nozzle section to be easily replaced or cleaned whilepermitting laminar flow. Referring to FIG. 4, it can be seen that theentire nozzle container (2) may be made of several components. Nozzlecontainer (2) may consist of cap section (23) to which piezoelectriccrystal (10) may be attached. Cap section (23) may be attached in somesealing fashion or may even be integral to nozzle body (24) as shown inFIG. 1. Similarly, nozzle body (24) may be sealingly attached to nozzletip (25). Each of these seals may consist of O-rings as but one exampleof the types of seals shown in FIG. 4. Nozzle tip (25) may be a ceramicfabricated item which includes an exit situated at its tip. This exitmay actually be an orifice made through techniques known by thoseskilled in the art (such as the use of tungsten wire and the like) so asto create a small orifice of about 50 to 150 microns in diameter.

Unlike the designs shown in the prior art such as those shown in FIG. 5,nozzle tip (25) need not be sealed to nozzle body (24) on its innersurface. Instead, the nozzle body inner surface (26) joins smoothly withthe nozzle tip inner surface (27) at tip joint (28). This smoothtransition is to the degree necessary to maintain laminar flow in theparticular application. It can be achieved through the inclusion of edgeinsert (29) within nozzle body (24) so as to allow nozzle tip (25) to beinserted into nozzle body (24). In this fashion seal (30) can bepositioned so as to contact the outer surface (31) of nozzle tip (25)and thus avoid any adverse impacts on laminar flow within nozzle volume(3). By locating seal (30) off of inner surface (27) of nozzle tip (25),the seal can be kept away from areas which are important to laminarflow. As may be understood, a great variety of designs may beaccomplished to achieve this goal. Importantly, it should be understoodthat inner surface (27) of nozzle tip (25) is defined merely withrespect to its function, namely, the surface which contacts and directsthe flow of sheath fluid of nozzle volume (3). Further, the definitionof "smooth" is also relatively defined as those transitions which do notsignificantly interrupt laminar flow and thus do not degrade theperformance of the flow cytometer. It should also be understood that theseal between any two components such as the seal between nozzle body(24) and nozzle tip (25) may be direct or indirect through the use ofintervening materials or components.

Yet another independent aspect of the invention is the aspect of beingable to adjust the location at which the substance is introduced. Asmentioned earlier, those skilled in the art have long recognized theneed to achieve variations in the entire process to accommodatevariations in conditions practically experienced. As shown in FIG. 3,the present invention affords the ability to vary the rate at whichsubstance is introduced without disrupting laminar flow and the like.This is achieved through positioning substance introduction port (9)within convergence zone (32) as may be easily understood and by varyingthe location of substance introduction port (9) within convergence zone(32). As shown, substance introduction port (9) may move along theprimary flow direction to maintain an optimal relationship to the flowof the sheath fluid. Through this technique, the relative concentrationsof the substance introduced and the sheath fluid can be varied. This canact to avoid the resolution drop and the like which the prior artappeared to consider unavoidable as they adapted to changing conditions.

Further, since it may be desirable to maintain equal velocities atsubstance introduction port (9), and since substance tube (33) may bemoved, it is possible to include a controller (34) which receivessignals from some type of sensor (14) and which may act to control amovement mechanism (35) and thus automatically adjust the location ofsubstance introduction port (9) within nozzle container (2). Further,controller (34) may act to additionally control the pressure ofsubstance reservoir (8) and sheath reservoir (5) for automaticcorrelation of the various factors based upon location or otherparameters sensed. Since the theoretical relationship between thesefactors is well known for optimal conditions and since the programmingor wiring of such a design could be easily achieved by those skilled inthe art, a variety of designs may be implemented to achieve this goal.Given the great variety of flow cytometer systems possible, it should beunderstood that a great variety of sensed values may be used rangingfrom concentration of the substance contained within substance reservoir(8), to the actual location of substance introduction port (9), to thepressure of the various sheath fluid or substance fluids, to some otherproperty of the substance sensed by sensor (14). Each of these--or anycombination of them and other factors--may be adjusted automatically toachieve desired relationships or to simply optimize results withoutregard to the actual predicted values. Naturally, in keeping with thisbroad concept it should be understood that sensor (14) may not be justone sensor but may in fact be a host of different sensors positioned atvarious locations depending upon the particular condition existingwithin the flow cytometer desired to be sensed. While, of course, thesensor (14) will only ascertain specific values, these values canindicate results which may be used to more appropriately adjust thelocation of the substance introduction port.

Similarly, a host of different designs for the location adjuster (shownin FIG. 3 as movement mechanism (35)) are possible. The locationadjuster may also include some type of screw means (36), that is, sometype of device which allows relatively continuous movement with fineadjustment. It may also include telescoping substance tube (37) (shownin FIG. 3 as potentially a redundant location adjuster for illustrativepurposes only) or perhaps some type of slide design through the capsection. In applications in which the conditions remain relativelystable, a replacement substance tube of fixed length may also beprovided. Thus, various substance tubes may be selected based upon theconditions encountered in that particular type of application. In thisfashion, the limitation experienced by the prior art whereby variationsin pressure were used but undesirably resulted in unequal fluidvelocities at the location of substance introduction port (9) can beavoided. This affords an increase in the resolution.

The foregoing discussion and the claims which follow describe thepreferred embodiment of the present invention. Particularly with respectto the claims and the broad concept discussed, it should be understoodthat changes may be made without departing from the essence of thispatented invention. It is intended that changes are permissible toaccommodate varying applications and will still fall within the scope ofthis patent. It is simply not practical to describe and claim allpossible revisions nor is it practical to claim all combinations of thevarying features. To the extent revisions utilize the essence of thepresent invention, each would naturally fall within the breath orprotection encompassed by this path. This is particularly true for thepresent invention since its basic concepts and understandings arefundamental in nature and can be broadly applied. It is alsoparticularly true since the present invention involves a number ofpotentially independent features which may be combined in synergisticways for particular applications.

I claim:
 1. A method of creating a droplet from a jet of a flowcytometer comprising the steps of:a. establishing a nozzle volumedefined by a nozzle body; b. introducing a flow of sheath fluid intosaid nozzle volume; c. introducing a flow of a substance within saidsheath fluid in said nozzle volume; d. establishing a substantiallyisolated unidirectional coupling with said nozzle volume which couplesan oscillator to said nozzle volume through use of a directionalisolator situated between said nozzle body and said oscillator; e.creating a substantially isolated unidirectional oscillation within saidnozzle volume using an alternating voltage with an amplitude of lessthan one hundred millivolts for said oscillator; f. allowing said sheathfluid to exit from said nozzle volume; and g. forming at least onesubstance entraining droplet from said sheath fluid after allowing saidsheath fluid to exit from said nozzle volume.
 2. A method of creating adroplet from a jet of a flow cytometer as described in claim 1 whereinthe amplitude of said alternating voltage is about ten millivolts.
 3. Amethod of creating a droplet from a jet of a flow cytometer as describedin claim 1 wherein said oscillator coupled to said nozzle volume throughthe use of a directional isolator situated between said nozzle body andsaid oscillator is established at least partially within said nozzlevolume.
 4. A method of creating a droplet from a jet of a flow cytometeras described in claim 1 wherein said nozzle volume has a perpendicularcross sectional area and wherein said oscillator is establishedsubstantially throughout said perpendicular cross sectional area.
 5. Amethod of creating a droplet from a jet of a flow cytometer as describedin claim 1 wherein said oscillator has an isolated unidirectionalcoupling to said sheath fluid.
 6. A method of creating a droplet from ajet of a flow cytometer as described in claim 1, 4, or 5 and furthercomprising the step of continuously converging said sheath fluid withinsaid nozzle volume.
 7. A system for creating a droplet from a jet of aflow cytometer comprising:a. a nozzle container establishing a nozzlevolume defined by a nozzle body and having a nozzle exit; b. a sheathfluid port located within said nozzle volume wherein said sheath fluidport introduces a sheath fluid; c. a substance introduction port locatedwithin said nozzle volume; d. an oscillator to which said sheath fluidis responsive; e. a substantially isolated unidirectional coupling whichcouples said oscillator to said nozzle volume through use of adirectional isolator situated between said nozzle body and saidoscillator wherein said coupling permits said oscillation to createoscillation in substantially one direction; f. an alternating voltagesource having an alternating voltage amplitude of less than one hundredmillivolts connected to said oscillator; and g. a free fall area belowsaid nozzle exit and within which a substance entraining droplet forms.8. A system for creating a droplet from a jet of a flow cytometer asdescribed in claim 7 wherein said alternating voltage amplitude is aboutten millivolts.
 9. A system for creating a droplet from a jet of a flowcytometer as described in claim 7 wherein said oscillator coupled tosaid nozzle volume through the use of a directional isolator situatedbetween said nozzle body and said oscillator is established at leastpartially within said nozzle container.
 10. A system for creating adroplet from a jet of a flow cytometer as described in claim 9 whereinsaid nozzle container has a cap section and wherein said oscillatorcomprises a piezoelectric crystal.
 11. A system for creating a dropletfrom a jet of a flow cytometer as described in claim 10 wherein saidsheath fluid port introduces a sheath fluid, wherein said oscillator hasan oscillator surface which faces said sheath fluid, and furthercomprising an interface material between said oscillator surface andsaid sheath fluid.
 12. A system for creating a droplet from a jet of aflow cytometer as described in claim 9 wherein said nozzle container hasa largest perpendicular cross sectional area and wherein said oscillatoris located at said largest perpendicular cross sectional area and issubstantially as large as said largest perpendicular cross sectionalarea.
 13. A system for creating a droplet from a jet of a flow cytometeras described in claim 10 wherein said directional isolator comprises aspacer.
 14. A system for creating a droplet from a jet of a flowcytometer as described in claim 7 or 12 wherein said nozzle containercontinuously converges.
 15. A system for creating a droplet from a jetof a flow cytometer as described in claim 14 wherein said convergingnozzle container continuously converges from said sheath fluid port tosaid nozzle exit.
 16. A system for creating a droplet from a jet of aflow cytometer as described in claim 14 wherein said converging nozzlecontainer comprises:a. a nozzle body having an inner surface; b. anozzle tip having an inner surface; and c. a seal located off of saidinner surface of said nozzle tip and to which both said nozzle body andsaid nozzle tip are responsive.
 17. A method of creating a droplet froma jet of a flow cytometer comprising the steps of:a. establishing anozzle volume defined by a nozzle body; b. introducing a flow of sheathfluid into said nozzle volume; c. introducing a flow of a substancewithin said sheath fluid in said nozzle volume; d. initiating asubstantially isolated unidirectional oscillation through use of adirectional isolator situated between said nozzle body and an oscillatorwherein said substantially unidirectional oscillation occurs within saidnozzle volume; e. allowing said sheath fluid to exit from said nozzlevolume; and f. forming at least one droplet from said sheath fluid afterallowing said sheath fluid to exit from said nozzle volume.
 18. A methodof creating a droplet from a jet of a flow cytometer as described inclaim 17 wherein said step of initiating substantially isolatedunidirectional oscillation through the use of a directional isolatorsituated between said nozzle body and an oscillator wherein saidsubstantially unidirectional oscillation occurs within said nozzlevolume comprises the step of establishing an isolated unidirectionalcoupling to said sheath fluid.
 19. A method of creating a droplet from ajet of a flow cytometer as described in claim 17 wherein saidsubstantially isolated unidirectional oscillation passes through amaterial interface and further comprising the step of minimizing thenumber of material interfaces which said substantially isolatedunidirectional oscillation must pass through.
 20. A method of creating adroplet from a jet of a flow cytometer as described in claim 19 whereinsaid substantially isolated unidirectional oscillation is created by anoscillator and wherein said substantially isolated unidirectionaloscillation passes through only a protective coating on said oscillatorbefore imparting on said sheath fluid.
 21. A method of creating adroplet from a jet of a flow cytometer as described in claim 19 whereinthe flow of said sheath fluid has a primary flow direction and whereinsaid step of minimizing the number of material interfaces which saidsubstantially isolated unidirectional oscillation must pass throughcomprises the step of coupling said substantially isolatedunidirectional oscillation in substantially only said primary flowdirection.
 22. A method of creating a droplet from a jet of a flowcytometer as described in claim 17 and further comprising the step ofdirectly transferring said substantially isolated unidirectionaloscillation to said sheath fluid.
 23. A method of creating a dropletfrom a jet of a flow cytometer as described in claim 17, 18, or 22 andfurther comprising the step of applying said substantially isolatedunidirectional oscillation to said sheath fluid.
 24. A method ofcreating a droplet from a jet of a flow cytometer as described in claim23 and further comprising the step of directionally isolating saidsubstantially isolated unidirectional oscillation with a spacer.
 25. Amethod of creating a droplet from a jet of a flow cytometer as describedin claim 17 and further comprising the step of directionally isolatingsaid substantially isolated unidirectional oscillation with a spacer.26. A method of creating a droplet from a jet of a flow cytometer asdescribed in claim 24 wherein said step of isolating said substantiallyisolated unidirectional oscillation comprises the step of coupling saidsubstantially isolated unidirectional oscillation to said sheath fluidalong only one plane.
 27. A system for creating a droplet from a jet ofa flow cytometer comprising:a. a nozzle container establishing a nozzlevolume defined by a nozzle body and having a nozzle exit; b. a sheathfluid port located within said nozzle volume wherein said sheath fluidport introduces a sheath fluid; c. a substance introduction port locatedwithin said nozzle volume; d. a substantially isolated unidirectionalcoupling which couples an oscillator to said nozzle volume through useof a directional isolator situated between said nozzle body and saidoscillator wherein said coupling permits said oscillator to createoscillation in substantially one direction; e. an oscillator to whichsaid substantially isolated unidirectional coupler and said nozzlevolume are responsive; and f. a free fall area below said nozzle exitand within which a substance entraining droplet forms.
 28. A system forcreating a droplet from a jet of a flow cytometer as described in claim27 wherein said sheath fluid port introduces a sheath fluid and whereinsaid oscillator comprises a piezoelectric crystal.
 29. A system forcreating a droplet from a jet of a flow cytometer as described in claim27 wherein said sheath fluid port introduces a sheath fluid, whereinsaid oscillator has an oscillator surface which faces said sheath fluid,and further comprising an interface material between said oscillatorsurface and said sheath fluid.
 30. A system for creating a droplet froma jet of a flow cytometer as described in claim 27 wherein said nozzlecontainer comprises:a. a cap section; b. a nozzle body sealed to saidcap section; and c. a nozzle tip having said nozzle exit situatedthereon, wherein said nozzle tip is sealed to said nozzle body, andwherein said sheath fluid flows through said nozzle tip.
 31. A systemfor creating a droplet from a jet of a flow cytometer as described inclaim 27 wherein said oscillator has an oscillator surface which facessaid sheath fluid and wherein said oscillator surface is planar.
 32. Asystem for creating a droplet from a jet of a flow cytometer asdescribed in claim 27 or 28 wherein said sheath fluid port introduces asheath fluid and further comprising a coupling which is only planar andwhich couples said oscillator to said sheath fluid.
 33. A system forcreating a droplet from a jet of a flow cytometer as described in claim32 wherein said directional isolator between said oscillator and saidnozzle body comprises a spacer.
 34. A system for creating a droplet froma jet of a flow cytometer as described in claim 27 wherein saidoscillator emits a predominant frequency and wherein said directionalisolator is effective at said predominant frequency.
 35. A system forcreating a droplet from a jet of a flow cytometer as described in claim33 wherein said oscillator has an oscillator side and wherein saidspacer maintains said oscillator side detached from said nozzlecontainer.